Achyranthes bidentata polysaccharides (ABPS) are the active components of Radix Achyranthis Bidentatae (AB), which has been extensively used in Traditional Chinese medicine (TCM) in the treatment of osteoarthritis (OA). Our previous study provided evidence that ABPS regulated the G1/S transition to promote chondrocyte proliferation. However, the precise mechanisms involved remain to be elucidated. In the present study, we aimed to investigate the effects of ABPS on the Wnt/β‑catenin signaling pathway in chondrocytes. Chondrocytes, obtained from the knee cartilage of Sprague-Dawley rats, were identified by type II collagen immunohistochemistry. ABPS upregulated the expression of Wnt-4, Frizzled-2, β-catenin and cyclin D1, and downregulated the expression of glycogen synthase kinase 3β (GSK-3β), as shown by reverse transcription PCR (RT-PCR) and western blot analysis. Using immunofluorescence, we also found that ABPS induced β-catenin nuclear translocation. Importantly, the expression of β-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/β-catenin signaling pathway. In addition, we found that ABPS increased the expression of type II collagen in chondrocytes. These results suggest that ABPS promote chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway.

Psoralen (PSO), the active ingredient of Fructus Psoraleae (FP) the dried ripe fruit of Psoralea corylifolia L., has been commonly used in traditional Chinese medicine (TCM) for the treatment of osteoarthritis (OA). We found that PSO activates cartilaginous cellular functions of rat chondrocytes in vitro. However, the effect of PSO on chondrocyte proliferation and the precise mechanisms involved remain to be elucidated. We investigated the effects of PSO on chondrocytes isolated from Sprague-Dawley (SD) rats and evaluated involvement of the Wnt/β-catenin signaling pathway. The viability of chondrocytes treated with PSO was increased in a dose- and time-dependent manner, as assessed by MTT assay. We found that the gene expression and protein levels of Wnt-4, Frizzled-2, β-catenin and cyclin D1 in the PSO-treated chondrocytes were significantly upregulated, while the gene expression and protein level of glycogen synthase kinase-3β (GSK-3β) were downregulated, compared with the untreated chondrocytes. By immunofluorescence, we also found that PSO induced β-catenin nuclear translocation. Importantly, the expression of β-catenin and cyclin D1 was partly inhibited by Dickkopf-1 (DKK-1), an inhibitor of the Wnt/β-catenin signaling pathway. Additionally, Col-II expression in chondrocytes was increased after treatment with PSO. Taken together, these results indicate that PSO promotes chondrocyte proliferation by activating the Wnt/β-catenin signaling pathway, and it may play an important role in the treatment of OA.

Chondrocyte apoptosis triggered by endoplasmic reticulum (ER) stress plays a vital role in the pathogenesis of osteoarthritis (OA). Bushen Zhuangjin decoction (BZD) has been widely used in the treatment of OA. However, the cellular and molecular mechanisms responsible for the inhibitory effects of BZD on chondrocyte apoptosis remain to be elucidated. In the present study, we investigated the effects of BZD on ER stress-induced chondrocyte apoptosis using a chondrocyte in vitro model of OA. Chondrocytes obtained from the articular cartilage of the knee joints of Sprague Dawley (SD) rats were detected by immunohistochemical staining for type II collagen. The ER stress-mediated apoptosis of tunicamycin (TM)-stimulated chondrocytes was detected using 4-phenylbutyric acid (4-PBA). We found that 4-PBA inhibited TM-induced chondrocyte apoptosis, which confirmed the successful induction of chondrocyte apoptosis. BZD enhanced the viability of the TM-stimulated chondrocytes in a dose- and time-dependent manner, as shown by MTT assay. The apoptotic rate and the loss of mitochondrial membrane potential (ΔΨm) of the TM-stimulated chondrocytes treated with BZD was markedly decreased compared with those of chondrocytes not treated with BZD, as shown by 4′,6-diamidino-2-phenylindole (DAPI) staining, Annexin V-FITC binding assay and JC-1 assay. To further elucidate the mechanisms responsible for the inhibitory effects of BZD on TM-induced chondrocyte apoptosis mediated by ER stress, the mRNA and protein expression levels of binding immunoglobulin protein (Bip), X-box binding protein 1 (Xbp1), activating transcription factor 4 (Atf4), C/EBP-homologous protein (Chop), caspase-9, caspase-3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were measured by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. In the TM-stimulated chondrocytes treated with BZD, the mRNA and protein expression levels of Bip, Atf4, Chop, caspase-9, caspase-3 and Bax were significantly decreased, whereas the mRNA and protein expression levels of Xbp1 and Bcl-2 were significantly increased compared with the TM-stimulated chondrocytes not treated with BZD. Additionally, all our findings demonstrated that there was no significant difference between the TM-stimulated chondrocytes treated with BZD and those treated with 4-PBA. Taken together, our results indicate that BZD inhibits TM-induced chondrocyte apoptosis mediated by ER stress. Thus, BZD may be a potential therapeutic agent for use in the treatment of OA.

Chondrocyte apoptosis activated by the mitochondrial-dependent signaling pathway plays a crucial role in the cartilage degeneration of osteoarthritis. Duhuo Jisheng decoction (DHJSD), a herbal formula from traditional Chinese medicine, has been widely used for treating osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effect of DHJSD remain to be elucidated. In the present study, the effects of DHJSD on the mitochondrial-dependent signaling pathway in sodium nitroprussiate (SNP)-induced chondrocyte apoptosis were investigated. Chondrocytes, from the knee articular cartilage of Sprague Dawley rats, were identified by type II collagen immunohistochemistry. The chondrocytes, stimulated with or without SNP to induce apoptosis, were treated by DHJSD for various concentrations and times. The viability of SNP-induced chondrocytes treated with DHJSD was enhanced compared to SNP-induced chondrocytes in a dose- and time-dependent manner, as assessed by the MTT assay. The apoptosis of SNP-induced chondrocytes treated by DHJSD was significantly decreased compared to SNP-induced chondrocyte, as shown by 4',6-diamidino-2-phenylindole and Annexin V/propidium iodide staining. The mitochondrial membrane potential (∆Ψm) of SNP-induced chondrocytes treated by DHJSD was significantly decreased compared to SNP-induced chondrocyte, as shown by JC-1 staining. To understand the mechanism, the mRNA and protein levels of Bax, B-cell lymphoma 2 (Bcl-2), caspase-9 and caspase-3 were detected by reverse transcription‑polymerase chain reaction and western blot analysis, respectively. In SNP-induced chondrocyte treated by DHJSD, the Bcl-2 expression was increased, whereas the expression of Bax, caspase-9 and caspase-3 was decreased compared to SNP-induced chondrocyte. Taken together, these results indicated that DHJSD inhibits the apoptosis of SNP-induced chondrocyte by the mitochondrial-dependent apoptotic pathway, and this may partly explain its therapeutic efficacy for OA.

The present study investigated the mechanism underlying the effects of glucosamine (GlcN) on the proliferation of chondrocytes isolated from the knee cartilage of Sprague-Dawley rats. Chondrocytes were treated with various concentrations of GlcN or without GlcN. The effects of GlcN on chondrocyte proliferation were determined using reverse transcription-polymerase chain reaction, western blot analysis and immunohistochemistry. The results indicated that GlcN significantly improved chondrocyte viability, accelerated G1/S transition during progression of the cell cycle and promoted the expression of cell cycle regulatory proteins, including cyclin D1, cyclin-dependent kinase (CDK)4 and CDK6, thus indicating that GlcN may promote chondrocyte proliferation. Furthermore, GlcN upregulated the expression levels of Wnt-4, Frizzled-2 and β-catenin, and downregulated the expression of glycogen synthase kinase-3. GlcN also promoted β-catenin translocation; β-catenin is able to activate numerous downstream target genes, including cyclin D1. To determine the role of the Wnt/β-catenin signaling pathway in chondrocyte proliferation, the Wnt/β-catenin signaling pathway was inhibited using Dickkopf-1 (DKK-1), after which chondrocytes were treated with GlcN. The results demonstrated that the expression levels of β-catenin and cyclin D1 were decreased in chondrocytes treated with DKK-1 and GlcN. These results suggested that GlcN may promote chondrocyte proliferation via the Wnt/β-catenin signaling pathway.

Bushen Zhuangjin Decoction (BZD), a well-known formulation in Traditional Chinese Medicine, has been widely used for the treatment of osteoarthritis (OA). Due to the poor intrinsic repair capacity of chondrocytes, promoting the proliferation of chondrocytes is an efficient treatment to delay the progression of cartilage degradation. The present study, therefore, focused on the effect of BZD on chondrocyte proliferation, exploring the mechanism of BZD on the inhibition of cartilage degradation. Chondrocytes isolated from the knee articular cartilage of Sprague Dawley rats were cultured and identified by type II collagen immunohistochemistry. It was found that BZD promoted chondrocyte viability in a dose- and time-dependent manner. To investigate if BZD promoted the chondrocyte viability by stimulating the cell cycle progression a flow cytometer was used, and the results showed that the percentage proportion of G0/G1 cells was significantly lower, and the percentage proportion of S cells was significantly higher, in treated cells compared with that in untreated cells. To gain insight into the mechanism underlying the effect of BZD on the cell cycle progression, the mRNA and protein expression of cyclin D1, cyclin-dependent kinase 4 (CDK4), CDK6 and p21 was measured by reverse transcription-polymerase chain reaction and western blotting, respectively. The mRNA and protein expression of cyclin D1, CDK4 and CDK6 in the BZD-treated chondrocytes was significantly upregulated, while the mRNA and protein expression of p21 was significantly downregulated, compared with that in the untreated chondrocytes. These results suggested that BZD promoted chondrocyte proliferation by accelerating G1/S transition, indicating that BZD is a potential therapeutic agent for the treatment of OA.

Abstract. Our previous study showed that Duhuo Jisheng decoction (DHJSD) inhibited chondrocyte apoptosis by the mitochondria-dependent signaling pathway. Endoplasmic reticulum (ER) stress is upstream of the mitochondria-dependent signaling pathway and has been shown to promote chondrocyte apoptosis that occurs in osteoarthritis (OA). The present study aimed to evaluate whether DHJSD inhibits the chondrocyte apoptosis by regulating ER stress. DHJSD enhanced the viability of tunicamycin (TM)-exposed chondrocytes, a model of ER stress-induced apoptosis, in a dose-and time-dependent manner, as shown by MTT assay. The present results showed that DHJSD and sodium 4-phenylbutyrate (PBA), an ER stress inhibitor, reduced TM-induced chondrocyte apoptosis by 4' ,6-diamidino-2-phenylindole staining. To gain insight into the mechanisms of DHJSD that are responsible for enhancing the viability and inhibiting TM-induced chondrocyte apoptosis, the associated mRNA expressions and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The results showed that the expression levels of Xbp1, Xbp1s and Bcl-2 were increased, and the expression levels of Bip, Atf4, Chop, Bax, caspase-9 and -3 were decreased in the TM-exposed chondrocytes treated with DHJSD or PBA compared with that in the TM-exposed chondrocytes. To identify the possible mechanisms, the expression of miR-34a was examined by the TaqMan microRNA assay, and was downregulated in the TM-exposed chondrocytes treated with DHJSD or PBA compared with that in the TM-exposed chondrocytes. DHJSD inhibits ER stress in chondrocytes induced by exposure to TM by downregulating miR-34a, suggesting that DHJSD may be a potential therapeutic agent for OA.

The role of short stature homeobox 2 (shox2) in the development and homeostasis of the temporomandibular joint (TMJ) has been well documented. Shox2 is known to be expressed in the progenitor cells and perichondrium of the developing condyle. A previous study by our group reported that overexpression of shox2 leads to congenital dysplasia of the TMJ via downregulation of the Indian hedgehog (Ihh) signaling pathway, which is essential for embryonic disc primordium formation and mandibular condylar growth. To determine whether overexpression of Ihh may rescue the overexpression of shox2 leading to congenital dysplasia of the TMJ, a mouse model in which Ihh and shox2 were overexpressed (Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice) was utilized to assess the consequences of this overexpression on TMJ development during post-natal life. The results showed that the developmental process and expression levels of runt-related transcription factor 2 and sex determining region Y-box 9 in the TMJ of the Wnt1-Cre; pMes-stop shox2; pMes-stop Ihh mice were similar to those in wild-type mice. Overexpression of Ihh rescued shox2 overexpression-associated reduction of extracellular matrix components. However, overexpression of Ihh did not inhibit the shox2 overexpression-associated increase of matrix metalloproteinases (MMPs) MMP9, MMP13 and apoptosis in the TMJ. These combinatory cellular and molecular defects appeared to account for the observed congenital dysplasia of TMJ, suggesting that overexpression of Ihh partially rescued shox2 overexpression-associated congenital dysplasia of the TMJ in mice.